During his FIRST award, Dr. Piletz (the P.I.) and colleagues obtained evidence that non-adrenergic, imidazoline-selective receptors may be linked to depression. They found that unipolar depressed patients displayed a significant elevation (p=.002) in the binding density (Bmax) of p-125 IC) to an imidazoline-selective site on platelets, compared to healthy controls or patients with generalized anxiety disorder. The site on platelet plasma membranes was nearly identical to the Imidazoline-1 Receptor (I1R) in brain stem based on its affinity for a variety of compounds. Platelet p125 IC binding declined in patients following antidepressant drug treatment. Additionally, the binding of an analogue, p-NH4-3H-clonidine, was significantly elevated in the late luteal phase of women with dysphoric premenstrual changes (PMS), but not in women with normal menstrual cycles. In this application Dr. Piletz teams up with the discoverer of the I1R (Dr. Ernsberger) to investigate the molecular basis of regulation of I1 binding sites. A human megakaryocytic tumor cell line (MEG-01) will be used as a model of platelet receptors. This unique cell line was shown by Dr. Piletz to possess I1 sites, and can actually produce platelet-like particles in culture. Drawing on Dr. Piletz's postdoctoral experience with cDNA, and on a team of colleagues who have cloned similar receptors, this application proposes to clone the I1 receptor cDNA in MEG-01 cells. These studies will answer fundamental questions about the platelet I1 site, its regulation in health and disease and its possible utility as a marker in depressive illness and PMS.